PROJECT SUMMARY Myocardial infarction (MI) alone causes 1 of every 7 deaths in the United States. As the heart has little regen- erative capacity after injury, there is an urgent need for novel therapeutic approaches to MI. Improving myo- cardial repair after MI will require enhancing vascular supply to regions with marginal perfusion and stimulating myocardial regeneration through formation of new cardiomyocytes and supporting vasculature. Endothelial-to- mesenchymal transition (EndoMT) plays a key role in embryonic development and in adult cardiovascular dis- eases (CVDs). While several studies have shown the importance of EndoMT in heart failure (HF), our overall knowledge of this process remains in its infancy in part due to the lack of mechanistic insight by which EndoMT genes are regulated and the absence of definitive studies using specific tools to establish the role of EndoMT in adult HF. The goal of this proposal is to leverage novel discoveries made by our group to define the role of a new microRNA (miR) regulatory mechanism of EndoMT in MI, with an ultimate view to clinical translation. Be- ta-arrestin (beta-arr)-biased beta2-adrenergic receptor (beta2AR) signaling in the heart elicits cardioprotection and may underlie the beneficial effects of the betaAR antagonist (beta-blocker) carvedilol (Carv). We recently discovered that Carv, acting through this mechanism, promotes the maturation of miR-532-5p (miR-532), which plays a vital cardioprotective role. MiR-532 induces cardiac endothelial cell (CEC) function and neovas- cularization after MI, and represses protease serine 23 (prss23; a key initiator of EndoMT, and a direct and functional target of miR-532 in CECs). Interestingly, the expression of miR-532 is significantly enriched in CECs and selectively downregulated in CECs isolated from ischemic myocardium, which is inversely associat- ed with the expression of prss23. Thus, a greater understanding of miR-532-mediated cardiac protection, and establishing the importance of increased miR-532 to the outcome of EndoMT and cardiac function might lead to the development of novel effective therapies (eg. beta2AR-selective beta-arr biased ligands) for numerous CVDs. Our hypothesis is that beta2AR/beta-arr-mediated activation of miR-532 maturation and function in ECs results in beneficial adaptive remodeling in infarcted hearts by repressing prss23 and EndoMT. To test our hy- pothesis, we plan to pursue three aims: 1) determine the contribution of miR-532 expression in ECs to post- ischemic heart remodeling, 2) elucidate the role of prss23, a novel direct and functional CEC target of miR-532 in MI, and (3) investigate the impact of beta-arr-biased agonism of beta2AR on CEC and heart function modu- lated by the miR-532/prss23 axis. This proposal is innovative because the role of the beta2AR/beta-arr/miR- 532/prss23 axis in EndoMT-related cardiac pathologies has never been studied, and proposed EC-specific miR-532 conditional knockout (KO) and prss23 KO mouse models are entirely novel. This project is also signif- icant because the crosstalk between beta2AR/beta-arr signaling and miR-532/prss23 axis can be exploited as a novel target for studying underlying mechanisms and treatment of organ fibrosis mediated by EndoMT.